Mutant alleles of myosin XVA cause profound congenital deafness DFNB3. In humans worldwide, mutations of MYO15A are a common cause of nonsyndromic deafness (Rehman et al., 2016). Myosins are actin-activated molecular motors that have a conserved head (motor) and neck (light chain binding motifs) and highly divergent tail domains. The tail of MYO15A has several motifs that are candidates for protein interaction motifs. To date, isoform 1 of myosin XVa is the largest of all reported vertebrate myosins ( 400 kDa). The N-terminus of myosin XVa is composed of 1,220 residues and encoded by a single exon. We previously demonstrated that isoform 1 is necessary for hearing (Nal et al., 2007). As a collaboration with Drs. Sally Camper and Gregory Frolenkov, a mouse model has been developed that has a defective amino terminus which recapitulates the human phenotype (Fang et al., 2015). The identification of proteins that functionally interact with MYO15 may provide a means of determining the various roles of MYO15A in the auditory system. In addition, interacting proteins are themselves likely to play crucial functions in hearing and would be strong candidates for proteins encoded by other deafness loci. We are therefore using a yeast two hybrid system and MS screens to identify proteins that interact with the myosin XVA. Genes that encode poteins that interact with myosin XVA from these two screens will be further examined for biological relevance. Whirlin and EPS8 are reported partners of the tail domain of myosin XVa and are necessary for stereocilia elongation and staircase formation (Belyantseva et al. 2005). As a collaboration with Dr. Sellers (NHLBI), we are continuing to characterize the biophysical properties of myosin XVa (Bird et al., 2014). The function of additional novel isoforms of MYO15A are also being investigated (Rehman et al., 2016). MYO15A appears to be a master regulator of stereocilia architecture as well as necessary for the lifelong of maintenance of stereocilia. In addition, we have been studying mouse models that have abnormal stereocilia rootlets and are deaf. For example,TRIOBP-4 and TRIOBP-5 are necessary for rootlet formation and maintenace. Rootlets provide bundle stiffness necessary for the detection of sound. Different splice isoforms of TRIOBP are necessary for building rootlets while others are necessary for maintaining rootlets longterm.